Microglia Activate Migration of Glioma Cells through a Pyk2 Intracellular Pathway.

Glioblastoma is one of the most aggressive and fatal brain cancers due to the highly invasive nature of glioma cells. Microglia infiltrate most glioma tumors and, therefore, make up an important component of the glioma microenvironment. In the tumor environment, microglia release factors that lead to the degradation of the extracellular matrix and stimulate signaling pathways to promote glioma cell invasion. In the present study, we demonstrated that microglia can promote glioma migration through a mechanism independent of extracellular matrix degradation. Using western blot analysis, we found upregulation of proline rich tyrosine kinase 2 (Pyk2) protein phosphorylated at Tyr579/580 in glioma cells treated with microglia conditioned medium. This upregulation occurred in rodent C6 and GL261 as well as in human glioma cell lines with varying levels of invasiveness (U-87MG, A172, and HS683). siRNA knock-down of Pyk2 protein and pharmacological blockade by the Pyk2/focal-adhesion kinase (FAK) inhibitor PF-562,271 reversed the stimulatory effect of microglia on glioma migration in all cell lines. A lower concentration of PF-562,271 that selectively inhibits FAK, but not Pyk2, did not have any effect on glioma cell migration. Moreover, with the use of the CD11b-HSVTK microglia ablation mouse model we demonstrated that elimination of microglia in the implanted tumors (GL261 glioma cells were used for brain implantation) by the local in-tumor administration of Ganciclovir, significantly reduced the phosphorylation of Pyk2 at Tyr579/580 in implanted tumor cells. Taken together, these data indicate that microglial cells activate glioma cell migration/dispersal through the pro-migratory Pyk2 signaling pathway in glioma cells.

Glioblastoma development in mouse brain: general reduction of OCTs and mislocalization of OCT3 transporter and subsequent uptake of ASP+ substrate to the nuclei.

Organic cation transporters (OCTs) were first found and then isolated from cultured glioma cells. When glioma cells are implanted into brain the fate of OCTs varies with time after implantation and transporter type. Here we report that OCT1, OCT2 and OCT3 immunofluorescence is significantly reduced over time in implanted GL261 glioma cells, during tumor development in the brain. By day 21 after glioma implantation, OCT1, OCT2 and OCT3 immunofluorescence was reduced more than 10-fold in the cytoplasm of glioma cells, while OCT3 immunofluorescence became concentrated in the nucleus. The well-known fluorescent substrate for OCT transporters, 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+), previously shown to accumulate in glioma-cell cytoplasm in in vivo slices, began to accumulate in the nucleus of these cells, but not in cytoplasm, after 21 days post-implantation. Considering this mislocalization phenomenon, and other literature on similar nuclear mislocalization of different transporters, receptors and channels in glioma cells, we suggest that it is one of the "omens" preceding the motility and aggressivity changes in glioma behavior.

Visualization of implanted GL261 glioma cells in living mouse brain slices using fluorescent 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+).

Here we describe a new method of glioma cell visualization in living brain slices that can be used for evaluation of tumor size or visualization of internal tumor structures. Glial cells, as well as glioma cells of glial origin, express high levels of organic cation transporters. We demonstrate that application of a fluorescent substrate for these transporters 4-(4-(dimethylamino)-styryl)-N-methylpyridinium iodide (ASP+) to the incubation medium leads to quick accumulation of fluorescence in glioma cells during early developmental stages and in astrocytes, but not in neurons. Stained brain slices can be immediately investigated using confocal or fluorescence microscopy. Glioma and glial cells can be discriminated from each other because of their different morphology. The method described has the advantage of staining living tissue and is simple to perform.